In the field of functionalized nanoparticles one of the challenges is to easily and reliably attach peptides and proteins to larger scaffolds. Targeted nanoparticles require effective ligands and unconjugated peptides themselves are weakly immunogenic. Bioconjugate chemistry has provided a range of strategies, but most nanoparticulate conjugations suffer from limitations relating to one or more of the following: 1) low conjugation yields and necessitated purification steps; 2) incompatibility with biological buffers, making labeling of intact nanoparticles impossible; 3) variable labeling sites and conjugated polypeptide conformations, creating an inhomogeneous particle population of varying efficacy; 4) necessity for complex and exogenous chemical approaches.
Standard approaches for ligand attachment to aqueous nanoparticles make use of maleimides, succinimidyl esters and carbodiimide-activated carboxylic acids. These can covalently react with amine and thiol groups of polypeptides. The use of maleimide-lipids has been explored extensively for antibody-conjugated immunoliposomes. Conjugation yields may reach as high as 90% from an overnight reaction, but subsequent quenching of free maleimide groups and additional purification is required. Proteins may require a preparative step of thiolation and purification prior to conjugation. Antibody orientation is a major factor influencing the conjugated antibody target binding efficacy, but these approaches result in numerous antibody labeling sites and indiscriminate orientations. Biorthogonal synthetic strategies such as the click reaction have recently been applied to pre-formed liposomes, however these require the use exogenous catalysts and unconventional amino acids.
Another approach that is suitable for smaller peptides which are less prone to permanent denaturation in organic solvents is to conjugate the peptides to a lipid anchor. The resulting lipopeptides can then be incorporated along with the other lipids during the liposome formation process. This approach has been used to generate synthetic vaccines that induce antibody production against otherwise non-immunogenic peptides. However, due to their amphipathic character, in that case the lipopeptides were difficult to purify, with a yield of 5-10%. It has also been shown that lipopeptides do not fully incorporate into liposomes during the formation process, resulting in aggregation.